Ribosome-inactivating-proteins (RIPs) are plant proteins that catalytically inactivate eukaryotic ribosomes. RIPS have been shown to inactivate ribosomes by interfering with the protein elongation step of protein synthesis. For example, the RIP saporin (SAP) has been shown to inactivate 60S ribosomes by cleavage of the n-glycosidic bond of the adenine at position 4324 in the rat 28S ribosomal RNA (rRNA). This particular region in which A.sup.4324 is located in the rRNA is highly conserved among prokaryotes and eukaryotes. A.sup.4324 in 28S rRNA corresponds to A.sup.2660 in Escherichia coli (E. coli) 23S rRNA. Several RIP's also appear to interfere with protein synthesis in prokaryotes, such as E. coli. Since RIPs are toxic to eukaryotic cells and some RIPs are toxic to prokaryotes (see, e.g., Habuka et al. (1990) J. Biol. Chem. 265:10988-10992), they are difficult to express using recombinant DNA methodologies.
Several structurally related RIP's have been isolated from seeds and leaves of the plant Saponaria officinalis (soapwort). Among these, saporin-6 is the most active and abundant, representing 7% of total seed proteins. Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents, such as SDS, and a variety of proteases. The amino acid sequences of several saporin-6 isoforms from seeds are known and there appear to be families of saporin RIPs differing in few amino acid residues.
Because saporin is a type I RIP, it does not possess a cell-binding chain, like the toxins ricin and abrin. Consequently, its toxicity to whole cells is much lower than the toxins. When targeted to cells so that it is internalized by the cells, however, its cytotoxicity is 100- to 1000-fold more potent than ricin A chain. Because of its cytotoxicity, saporin has been covalently linked to cell surface binding ligands to produce cytotoxic chemical-conjugates or linked to antibodies to produce immunotoxins that are targeted to, and internalized by, specific cells (see, e.g., Soria (1989) Pharmacological Res. 21(Supp 2):35-46, at 36). For example, basic fibroblast growth factor (bFGF) has been chemically conjugated to saporin-6 to produce the mitoxin FGF-SAP (see, e.g., U.S. Pat. No. 5,191,067 to Lappi et al.; and Lappi et al. (1989) Biochem. and Biophys. Res. Comm. 160:917-923). FGF-saporin conjugates have been used to treat restinosis (see, e.g., International Patent Application No. WO 92/11872, which is based in U.S. application Ser. No. 07/637,074). Treatment is effected by local or intravenous administration of a therapeutically effective amount of the FGF conjugate following, for example, balloon angioplasty. FGF-saporin conjugates also have shown promise as agents for the treatment of certain tumors. The growth of melanomas and other tumors that express receptors to which FGF binds can be inhibited by FGF-SAP (see, e.g., International Application No. WO 92/04918, which is based on U.S. patent application Ser. No. 07/585,319: and Beitz et al. (1992) Cancer Research 52:227-230).
An anti-human immunoglobulin heavy chain monoclonal antibody has been conjugated to saporin-6. The resulting immunotoxin is potentially useful for eliminating lymphoma and leukemia cells from human bone marrow during ex vivo treatment prior to reimplantation. Other chemical conjugates of saporin with a panel of anti-T lymphocyte monoclonal antibodies have shown promise as ex vivo agents for purging human bone marrow prior to transplantation, and as systemic therapeutic agents in patients with graft-versus-host disease and T-cell and B-cell leukemia.
Presently, conjugation of saporin to cell binding ligands and antibodies has been effected chemically. Chemical conjugation, however, results in a heterogeneous population of molecules. For example, bFGF is conjugated via a cysteine residue to saporin, which is first derivatized with N-succinimdyl-3(2-pyridyldithio)propionate(SPDP). Basic FGF has at least two cysteines available for reaction with SPDP-derivatized saporin. Consequently, reaction of the bFGF with the SPDP-derivatized SAP results in an array of molecules, which probably differ with respect to biologically relevant properties and may not be ideal for in vivo applications.
In view of the many potential applications for saporin-containing fusion proteins, efficient recombinant means for the direct production of uniform preparations of saporin-containing proteins would be of great value. Because of the toxic effect of saporin on E. coli, as well as eukaryotes, recombinant production of biologically active saporin has, thus far, been elusive. DNA encoding saporin-6 has been cloned and a DNA encoding truncated form expressed in E. coli (U.K. Patent Application GB 2216891 A to FARMITALIA). The resulting protein, however, is not cytotoxic. DNA encoding recombinant bFGF-saporin fusion proteins in which the saporin and FGF are truncated have been prepared (see, Prieto et al. (1991) Ann. N.Y. Acad. Sci. 538:434-437). The resulting fusion protein, however, was subsequently found not to be cytotoxic.
Therefore, it is an object herein to provide effective recombinant DNA methods for the production of cytotoxic saporin-containing proteins, including fusion proteins, in prokaryotic cells. It is also an object herein to provide bFGF-SAP conjugates that are produced by recombinant DNA methods.